Evaluation of Metaplastic Triple-Negative Breast Cancer Extracellular Matrix Structure and Protein Composition

Alterations in the tumor extracellular environment and matrix stiffness promotes tumor progression. Furthermore, correlational studies have identified enrichment of extracellular matrix (ECM) proteins (glycoproteins, collagens) in breast tumors. Despite these findings, there has yet to be an interdisciplinary analysis of both ECM composition and structural architecture in rare breast tumors, such as metaplastic breast cancer. Here, we explored changes in ECM protein expression and architecture in a triple-negative breast cancer (TNBC) metaplastic tumor through SEM, proteomics, and RNA sequencing. SEM revealed tumor pore size was larger compared to control adipose tissue. Oscillating rheometry demonstrated increased ECM stiffness in the tumor compared to control adipose breast adipose. Proteomic analysis of the metaplastic TNBC tumor showed significant enrichment for ECM proteins, notably glycoproteins compared to control adipose. Interestingly, these samples showed no observed changes in expression for major fibrillar collagens COL1A1 and COL1A2, and a reduced expression of COL3A1. To determine the impact of less characterized ECMs in metaplastic TNBC, we overexpressed MFAP2 in primary metaplastic breast cancer cells and performed RNA sequencing. MFAP2 overexpression was associated with upregulation of epithelial-to-mesenchymal transition related genes . Overall, our results establish an extracellular signature and onco-architecture for the metaplastic triple-negative tumor type. Overall design: Metaplastic TNBC (TU-BcX-4IC) cell line constructed to overexpress the matrix protein MFAP2 or control vector. Overexpression (OE) was chieved through the transfected with 5 µg of pCMV6-Entry Mammalian Expression Vector DNA (Origene, MA, USA) and MAGP1 5ug (MFAP2) (NM_017459) Human Tagged ORF Clone (Origene, MA, USA). Following stable selection of MFAP2 overexpressing cell lines, RNA sequencing was performed. For RNA sequencing, metaplastic TU-BcX-4IC and TNBC cell line with pCMV6 Vector or MFAP2 OE cells were cultured in a T75 flask (Thermo Scientific, Waltham, Massachusetts, USA) grown to 80% confluency and collected for RNA extraction.